primers stopped working - (Jan/05/2007 )

Hi everyone

I am in need of some help with this weird problem i've been having.
The story is this: I ordered 4 pairs of primers. At first all of them worked, some better than others, but nothing that could not be dealt with by adjusting MgCl2 and adding DMSO to some of the rections.

Some time later (maybe a month, i dunno...), the 4th pair of primers stopped working with no apparent reason whatsoever. I tried everything I and my colleagues could remember:

- New primer dilutions from the stocks
- Various annealing temperatures
- Variations in Mg, buffer, DMSO and DNA concentration
- New solutions of everything (buffer, primer, water (milli-q), taq, dNTPs, Mg)
- Different termocyclers
- etc

Now, it could be possible that these primers had gone bad for some reason, but now comes the weird part: when I tried to combine these primers with others (for example primer F3 + primer R4), they worked! So they are functional but they don't work together (although they did at first).

To make things worse, about a month or two after all this, the exact same thing happened to the 3rd pair of primers. Again without any apparent reason. I did not change a thing from what I had been doing. And again, although they did not work together, they do when I combine them with other primers (again the example of primer F3 + primer R4, and so on).

I am kind of desperate, so I would like to know if anyone has any advice on this. I will probably order new primers so I can continue my work, because I am kind of stalled...

Thanks in advance

If any goes wrong with a PCR reaction (especially one that was working before) it is often the case that the template which is the cause of the problem. ie the template has degraded with time.

So with this rule of the thumb, could you look at your template? Has it begun to degrade? Meaning that the DNA has fragmented to such a degree that large PCR products can no longer be made.

[Hi
i too face the same trouble. but then i found that my DNA template was degrading with time. now am preparing DNA again. so why dont u check up the quality of ur template.
all the best


madhan shankar
Institute of Forest Genetics and Tree Breeding
India

That does make sense, I haven't checked the integrity of the genomic DNA I've been working with for a couple of months, but nevertheless the same thing happens when I try to amplify my fragments from a plasmid. Where I should get a single lane I get only smear.

I kind of developed a way of getting the work done by combining different primers, so I think I'll manage. Just wished I knew what's wrong with these godforsaken primers because it would make my work much easier.

Thanks for your replies you guys